There is a growing demand for binding molecules consisting of amino acids which are not immunoglobulins. While until now antibodies represent the best-established class of binding molecules there is still a need for new binding molecules in order to target ligands with high affinity and specificity since immunoglobulin molecules suffer from major drawbacks. Although they can be produced quite easily and may be directed to almost any target, they have a quite complex molecular structure. There is an ongoing need to substitute antibodies by smaller molecules which can be handled in an easy way. These alternative binding agents can be beneficially used for instance in the medical fields of diagnosis, prophylaxis and treatment of diseases.
Proteins having relatively defined 3-dimensional structures, commonly referred to as protein scaffolds, may be used as starting material for the design of said alternative binding agents. These scaffolds typically contain one or more regions which are amenable to specific or random sequence variation, and such sequence randomisation is often carried out to produce a library of proteins from which the specific binding molecules may be selected. Molecules with a smaller size than antibodies and a comparable or even better affinity towards a target antigen are expected to be superior to antibodies in terms of pharmacokinetic properties and immunogenicity.
A number of previous approaches do use protein scaffolds as starting material of binding proteins. For example, in WO 99/16873 modified proteins of the lipocalin family (so-called Anticalins) exhibiting binding activity for certain ligands were developed. The structure of peptides of the lipocalin family is modified by amino acid replacements in their natural ligand binding pocket using genetic engineering methods. Like immunoglobulins, the Anticalins can be used to identify or bind molecular structures. In a manner analogously to antibodies, flexible loop structures are modified; these modifications enable the recognition of ligands different from the natural ones.
WO 01/04144 describes the artificial generation of a binding domain on the protein surface. in beta sheet structural proteins per se lacking a binding site, By means of this de novo generated artificial binding domain e.g. variations in γ-crystallin—an eye lens structural protein—can be obtained which interact with ligands with high affinity and specificity. In contrast to the modification of binding sites which are already present and formed from flexible loop structures as mentioned above for Anticalins, these binding domains are generated de novo on the surface of beta sheets. However, WO 01/04144 only describes the alteration of relatively large proteins for the generation of novel binding properties. Due to their size the proteins according to WO 01/04144 can be modified on the genetic engineering level only by methods which require some effort. Furthermore, in the proteins disclosed so far only a relatively small proportion by percentage of the total amino acids was modified in order to maintain the overall structure of the protein. Therefore, only a relatively small region of the protein surface is available which can be utilized for the generation of binding properties that did not exist previously. Moreover, WO 01/04144 discloses only the generation of a binding property to γ-crystallin.
WO 04/106368 describes the generation of artificial binding proteins on the basis of ubiquitin proteins. Ubiquitin is a small, monomeric, and cytosolic protein which is highly conserved in sequence and is present in all known eukaryotic cells from protozoans to vertebrates. In the organism, it plays a crucial role in the regulation of the controlled degradation of cellular proteins. For this purpose, the proteins destined for degradation are covalently linked to ubiquitin or polyubiquitin chains during their passage through a cascade of enzymes and are selectively degraded because of this label. According to recent results, ubiquitin or the labelling of proteins by ubiquitin, respectively, plays an important role also in other cellular processes such as the import of several proteins or the gene regulation thereof.
Besides the clarification of its physiological function, ubiquitin is a research object primarily because of its structural and protein-chemical properties. The polypeptide chain of ubiquitin consists of 76 amino acids folded in an extraordinarily compact α/β structure (Vijay-Kumar, 1987): almost 87% of the polypeptide chain is involved in the formation of the secondary structural elements by means of hydrogen bonds. Prominent secondary structures are three and a half alpha-helical turns as well as an antiparallel β sheet consisting of four strands. The characteristic arrangement of these elements—an antiparallel β sheet exposed of the protein surface onto the back side of which an alpha helix is packed which lies vertically on top of it—is generally considered as so-called ubiquitin-like folding motif. A further structural feature is a marked hydrophobic region in the protein interior between the alpha helix and the β sheet.
Because of its small size, artificial preparation of ubiquitin can be carried out both by chemical synthesis and by means of biotechnological methods. Due to the favourable folding properties, ubiquitin can be produced by genetic engineering using microorganisms such as Escherichia coli in relatively large amounts either in the cytosol or in the periplasmic space. Because of the oxidizing conditions predominating in the periplasm the latter strategy generally is reserved for the production of secretory proteins. Due to the simple and efficient bacterial preparation ubiquitin can be used as a fusion partner for other foreign proteins to be prepared for which the production is problematic. By means of fusion to ubiquitin an improved solubility and thereby an improved production yield can be achieved.
Compared to antibodies or other alternative scaffolds, artificial binding proteins on the basis of ubiquitin proteins (also referred to as Affilin®) have the advantages of a small size and high stability, high affinity, high specificity, cost effective microbial manufacturing, and adjustment of serum half life. However, there is still a need to further develop those proteins in terms of immunogenic potential, fast and predictive preclinical development track and new therapeutic approaches. While WO 05/05730 generally describes the use of ubiquitin scaffolds in order to obtain artificial binding proteins, no solution is provided on how to modify and on how to efficiently select such a modified ubiquitin protein in order to obtain an even higher and more specific affinity binding to ligands like haptens and antigens, e.g. proteins and peptides and epitopes thereof.
The methods described in WO 05/05730 refer to monomers of modified ubiquitin proteins or to coupled proteins of modified ubiquitin. The coupled forms are generated by screening and selecting one, two or more modified ubiquitin proteins and combining them afterwards either by genetic or chemical methods to obtain coupled forms which enable for example multispecific binding of different kinds of ligands by one coupled ubiquitin molecule. In one example, site-directed coupling of two identical ubiquitin-based proteins (homo-dimers) is described in order to increase the binding affinity compared to a single modified ubiquitin molecule
It is an object of the present invention to provide a method on how to identify multimeric ubiquitin proteins with high binding capability to a ligand. It is a further object of the present invention to provide a method for identifying new binding proteins based on modified ubiquitin being able to bind specifically with high affinity to selected ligands.
The above-described objects are solved by the subject-matter of the enclosed independent claims. Preferred embodiments of the invention are included in the dependent claims as well as in the following description, examples and figures.